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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Mol Cell Endocrinol. Author manuscript; available in PMC Oct 26, 2009.
Published in final edited form as:
PMCID: PMC2767333
NIHMSID: NIHMS148501

Activity and intracellular location of estrogen receptors α and β in human bronchial epithelial cells

Abstract

Gender differences in lung disease and cancer are well-established. We reported estrogenic transcriptional responses in lung adenocarcinoma cells from females but not males despite similar estrogen receptor (ER) expression. Here we tested the hypothesis that normal human bronchial epithelial cells (HBECs) show gender-independent estrogenic responses. We report that a small sample of HBECs express ~twice as much ERβ as ERα.ERα and ERβ were located in the cytoplasm, nucleus, and mitochondria. In contrast to lung adenocarcinoma cells, estradiol (E2) induced estrogen response element (ERE)-mediated luciferase reporter activity in transiently transfected HBECs regardless of donor gender. Overexpression of ERα-VP16 increased ERE-mediated transcriptional activity in all HBECs. E2 increased and 4-hydroxytamoxifen and ICI 182,780 inhibited HBEC proliferation and cyclin D1 expression in a cell line-specific manner. In conclusion, the response of HBECs to ER ligands is gender-independent suggesting that estrogenic sensitivity may be acquired during lung carcinogenesis.

Keywords: Estrogen receptor, Lung adenocarcinoma (human), Normal lung (human), SMRT, Cyclin D1

1. Introduction

Gender differences in lung function and in lung disease indicate roles for estrogens and androgens in lung physiology (Carey et al., 2007a,b; Lim and Kobzik, 2008). Studies in rodents have revealed that estrogen receptors α and β (ERα and ERβ) play roles in normal lung function and pathophysiological conditions in lung (Carey et al., 2007a,b; Couse et al., 1997; Kuiper et al., 1997; Massaro et al., 2007; Massaro and DeCarlo Massaro, 2007; Massaro and Massaro, 2004, 2006; Matsubara et al., 2008; Morani et al., 2006; Patrone et al., 2003). Studies in mice in which ERα and/or ERβ were deleted (αERKO and βERKO) revealed that both ER subtypes are required for the formation of a full complement of alveoli in female mice, that ERα mediates the sexual dimorphism of body mass-specific alveolar number and surface area, and that absence of ERβ diminishes lung elastic tissue recoil (Massaro and Massaro, 2004, 2006). In humans, pre-pubescent boys have a higher incidence of asthma than girls, but after puberty, women have a higher incidence and worse symptoms than men, an observation that is likely related to the effect of estrogens on immune as well as lung functions (Almqvist et al., 2008). Indeed, postmenopausal use of hormone replacement therapy (HRT, estrogen plus progestin) and estrogen replacement therapy (ERT) was associated with a 2.29-fold higher risk for developing asthma, but not with development of chronic obstructive pulmonary disease (COPD) (Barr et al., 2004). Studies indicate that women are at higher risk for developing COPD (Han et al., 2007; Machado, 2008; Machado et al., 2006; Silverman et al., 2000). These studies, and numerous others, illustrate a role for gender, and likely sex steroids, in lung disease (Carey et al., 2007a,b), as well as the continued need for further study (Machado, 2008).

Lung cancer is the most common cancer in both men and women in the USA; however, although the incidence of lung cancer is declining in men, it is only now plateauing in women after rising for many decades (Jemal et al., 2007). Women are more likely than men to have lung adenocarcinoma, a histologic non-small cell lung cancer (NSCLC) subtype that correlates with worsened prognosis, but women have improved survival compared with men (Schwartz et al., 2005). The higher risk of lung adenocarcinoma in women prompted investigation of the possible role of ER in lung cancer (Dougherty et al., 2006; Hershberger et al., 2005; Marquez-Garban et al., 2007; Pietras et al., 2005; Stabile et al., 2002, 2005; Weinberg et al., 2005). These reports revealed that ERβ appears to be more highly expressed in lung cancer cells than ERα and that estrogen treatment activates both genomic (transcriptional) and non-genomic, i.e., membrane-initiated intra-cellular ERK1/ERK2 (MAPK) signaling, in lung cancer cells. A new clinical case study reported that 73% of 59 NSCLC patient samples examined had higher estradiol (E2) concentration in the carcinomas compared to the corresponding non-neoplastic lung tissues from the same patient, regardless of gender (Niikawa et al., 2008). Intratumoral E2 concentrations correlated with aromatase and PCNA mRNA, but not ERα or ERβ staining (Ishibashi et al., 2005). Thus, local estrogen production may play a role in NSCLC. We previously reported that both ERα and ERβ are expressed in human lung adenocarcinoma cell lines, normal lung fibroblasts, and normal human bronchial epithelial cells (HBECs) (Dougherty et al., 2006). However, only the lung adenocarcinoma cell lines derived from female patients responded proliferatively and transcriptionally to estradiol (E2) and other ER ligands (Dougherty et al., 2006). The subcellular location and activity of ERα and ERβ in HBECs has not been investigated.

In this study, we examined the expression and activity of ERα and ERβ in HBECs immortalized with Cdk4 and human telomerase reverse transcriptase (hTERT) (Ramirez et al., 2004). Microarray studies showed that these HBEC cell lines have similar gene expression signatures that were distinct from those of lung cancer cell lines and were comparable to those from primary HBECs (Ramirez et al., 2004). To address whether the differences in estrogenic responses that we detected between lung adenocarcinoma cell lines from female versus male lung adenocarcinoma patients (Dougherty et al., 2006) was present in HBECs, we evaluated ERα and ERβ expression, intracellular location, and function in HBECs derived from female and male patients. We describe differences in estrogen and antiestrogen responses between HBECs and lung adenocarcinoma cells. As shown in the present report, there were no gender-dependent differences in the ER responses in HBECs, thus differentiating HBECs from lung adenocarcinoma cell lines.

2. Materials and methods

2.1. Chemicals

17β-estradiol (E2) and 4-hydroxytamoxifen (4-OHT) were purchased from Sigma–Aldrich (St. Louis, MO). ICI 182,780 was purchased from Tocris (Ellisville, MO).

2.2. Antibodies

Antibodies were purchased as follows: ERα (HC-20) and ERβ (H-150) from Santa Cruz Biotechnology (Santa Cruz, CA); ERα (AER320) and α-tubulin from NeoMarkers (Freemont, CA), ERβ (PA1-311) from Affinity BioReagents (Goldon, CO), ERβ (6A12, MS-ERB11-PX2) from GeneTex (Austin, TX), SMRT monoclonal antibody from BD Biosciences Pharmingen (San Diego, CA), and β-actin from Sigma–Aldrich.

2.3. Cell culture and treatment

The properties of the 5 human bronchial epithelial cells cell lines prepared from 1 male and 2 female patients used in this study are provided in Table 1 and were originally described in (Ramirez et al., 2004). HBEC were maintained in keratinocyte-serum free medium supplemented with 2.5 μg recombinant human (rh) epidermal growth factor (EGF) and 25 mg bovine pituitary extract from Invitrogen (Carlsbad, CA). MCF-7 cells were purchased from ATCC (Manasas, VA) and maintained as described (Dougherty et al., 2006). Prior to treatment the cells were placed in phenol red-free media supplemented with 5% dextran-coated charcoal stripped FBS (DCCFBS) for 24–48 h. Ethanol (EtOH) was used as the vehicle control for all experiments. Cells were treated with various concentrations of E2 as indicated in the Figures. For the indicated experiments the cells were pretreated with 100 nM ICI 182,780 for 6 h prior to E2 treatment.

Table 1
Cell lines used in this study

2.4. Cell proliferation/Bromodeoxyudridine (BrdU) incorporation assays

For measuring cell proliferation, cells were plated in 96 well plates in keratinocyte-serum free medium for 24 h. Treatments (vehicle control, i.e., ethanol (EtOH), E2, 4-OHT, or ICI 182,780, alone or in combination) were added for 48 h. The BrdU ELISA assay from Roche Diagnostics (Indianapolis, IN) was performed according to the manufacturer’s instructions. Within each experiment, each treatment was performed in quadruplicate and values were averaged. Values were compared to those in the wells treated with vehicle (EtOH) control which was set to 100. At least 4 separate experiments were performed for each cell line.

2.5. Transient transfection

For transient transfection, cells were plated in 24-well plates at a density of 2.5 × 104 cells/well in keratinocyte-serum free medium supplemented with 2.5 μg recombinant human epidermal growth factor and 25 mg bovine pituitary extract (Invitrogen). Transient transfection was performed using FuGene6 (Roche) in phenol red-free OPTI-MEM I reduced serum medium (GIBCO/Invitrogen). Each well received 250 ng of a pGL3-pro-luciferase reporter (Promega, Madison, WI) containing 2 tandem copies of a consensus estrogen response element (i.e., EREc38 (Tyulmenkov et al., 2000)) and 5 ng of a Renilla luciferase reporter (pRL-tk) from Promega. For the indicated experiments, HBECs were also transfected with 10 ng of the expression vectors ERα-VP16, ERβ-VP16, and ERα-AF-1 (ERα-AF-1 has 3aa mutations in the LBD that preclude ligand binding (Tzukerman et al., 1994)) that were created by Dr. Donald McDonnell (Hall and McDonnell, 1999; McDonnell et al., 1995) and purchased from Addgene (Cambridge, MA). Prior to treatment the cells were placed in keratinocyte-serum free medium for 24 h. Forty-eight hours after transfection, triplicate wells were treated with EtOH (vehicle control), E2, 4-OHT, and ICI 182,780, as indicated in the Figures. The cells were harvested 30 h. post-treatment using Promega’s Passive Lysis buffer. Luciferase and Renilla luciferase activities were determined using Promega’s Dual Luciferase assay in a Plate Chameleon luminometer (BioScan, Washington, DC). Firefly luciferase was normalized by Renilla luciferase to correct for transfection efficiency. Fold induction was determined by dividing the averaged normalized values from each treatment by the EtOH value for each transfection condition within that experiment. Values were averaged from multiple experiments as indicated in the Figure legends.

2.6. Protein isolation

Whole cell extracts (WCE) were prepared in modified RIPA buffer as described in Dougherty et al. (2006). Protein concentrations were determined using the Bio-Rad DC Protein Assay (Bio-Rad Laboratories, Hercules, CA).

2.7. Western blotting

Western analysis was performed as described (Dougherty et al., 2006). Briefly, WCE were resolved by SDS-PAGE and electroblotted onto PVDF membranes (Pall Corporation, Pensacola, FL). Membranes were blocked in 5% dry milk TRIS-buffered saline (TBS) and probed with anti-ER primary antibodies. Membranes were stripped and re-probed for α-tubulin or β-actin for normalization. For SMRT westerns, 35 μg of WCE were separated on 3–8% Tris-Acetate NuPAGE gels (Invitrogen) prior to transfer to PVDF. To estimate ERα and ERβ protein expression, known concentrations, as assayed by [3H]E2 binding (Kulakosky et al., 2002), of baculovirus-expressed recombinant human ERα and ERβ protein were separated in parallel with the WCE of the HBECs and used as calibrators to estimate ERα and ERβ expression levels by Western blot (Figs. (Figs.11 and and22)(Dougherty et al., 2006).

Fig. 1
HBECs express ERα. WCE (40 μg) from the indicated cell lines and the indicated amounts of baculovirus-expressed ERα were separated by 10% SDS PAGE, transferred to PVDF membranes, and probed with AER320 monoclonal antibody for ERα ...
Fig. 2
HBECs express ERβ. WCE (40 μg for the PA1-311 and 6A12 blots and 30 μg for the H150 blot) from the indicated cell lines and the indicated amounts of baculovirus-expressed ERβ were separated by 10% SDS PAGE, transferred ...

2.8. RNA extraction and quantitative real time RT-PCR

RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. RNA quality was determined using the RNA 6000 Nano Assay kit on the Agilent 2100 Bioanalyzer (Wilmington, DE) by absorbance at 260 nm. 2 μgRNA was reverse transcribed using random hexamers and the High Capacity cDNA archive kit (PE Applied Biosystems (ABI), Foster City, CA). The QIAquick PCR purification kit (Qiagen) was used to purify cDNA.

Progesterone receptor (PR, PGR), cyclin D1 (CCND1), NRF-1 (NRF1), pS2 (TFF1) and 18S rRNA Taqman primers and probes were purchased as Assays-on-DemandTM Gene Expression Products (ABI). Each sample was normalized using 18S rRNA. Real-time PCR was performed in the ABI PRISM 7700 SDS 2.1 using relative quantification as described previously (Dougherty et al., 2006). Analysis and fold differences were determined using the comparative CT method as described in the ABI technical bulletin #2 (Bustin, 2002; Ginzinger, 2002). Fold change was calculated from the ΔΔCT values with the formula 2−ΔΔCT and are relative expression compared to vehicle control in each cell line.

2.9. Confocal laser scanning microscopy

Cells were plated on chamber slides (Lab-TekII, Nalge Nunc, Rochester, NY) for 48–72 h prior to incubation with 25 nM MitoTracker Red CMXRos (Molecular Probes, Eugene, OR) for 45–60 min. Mitotracker Red is oxidized in active mitochondria thus providing a functional assay of mitochondrial activity. Cells were then fixed with 1:1 methanol/acetone followed by incubation with ER primary antibodies (ERα: HC-20 or ERβ: H150, both from Santa Cruz) at a 1:300 dilution for 1 h. The secondary antibody was labeled with Quantum Dot 525 (Quantum Dot Corporation). Cells were then incubated with ProLong® Gold antifade reagent with DAPI (Molecular Probes) before analysis by confocal microscopy (Zeiss Axiovert100) using LSM510 software (Zeiss).

2.10. Statistics

Data are presented as mean ± SE. Statistical analyses were performed using Student’s t-test or one-way ANOVA followed by Student–Newman–Keuls or Dunnett’s post hoc tests using GraphPad Prism (San Diego, CA).

3. Results

3.1. HBECs express ERα and ERβ

The expression of ERα and ERβ proteins in WCEs from HBECs was investigated by western analysis. Standard amounts of baculovirus-expressed recombinant human ERα and ERβ were resolved in parallel and used to estimate ERα and ERβ concentrations (Dougherty et al., 2006). WCE from MCF-7 breast cancer cells served as an additional control for ERα and ERβ (Dougherty et al., 2006). Full length (67kDa) ERα was expressed in HBECs, H1395 lung adenocarcinoma cells and MCF-7 cells (Fig. 1A). The average expression of ERα in the HBECs ranged from 0.08 to 0.11 fmol/μg of protein (Fig. 1B). Concurrent analysis revealed that the expression of ERα in MCF-7 cells was 0.22 fmol/μg, i.e., ~twice that of the HBECs (Fig. 1B). Antibodies raised against the N-terminus of ERα also detected ERα in HBECs (Supplemental Fig. 1). Similar analyses revealed that ERβ expression ranged from 0.16 to 0.22 fmol/μg protein which was similar to the amount of ERβ expressed in MCF-7 cells (Fig. 2B). Uncropped images of the ERβ westerns are included as Supplemental Fig. 2. Thus, ERβ is ~2-fold higher than ERα in HBECs.

3.2. Subcellular localization of ERα and ERβ in HBECs

We examined the effect of E2 on the intracellular localization of ERα and ERβ in HBECs using immunohistochemistry (IHC) with antibodies unique for each ER subtype and confocal microscopy (Fig. 3, Supplemental Fig 3, and summarized in Table 2). DNA was stained by DAPI. Respiring mitochondria were specifically imaged by MitoTracker Red CMXRos. As a negative control, no green immunofluorescence was detected when the primary ERα or ERβ antibodies were excluded from the incubation, although MitoTracker and DAPI staining was appropriately detected (Supplemental Fig 4). A Western blot for ERα using the HC-20 ERα antibody is shown in Supplemental Fig 5. In all cells examined, MitoTracker signaling consistently indicated localization of the active mitochondria in the perinuclear region. In all HBECs, ERα and ERβ were predominantly cytoplasmic but were also present in the nucleus in both untreated and E2-treated cells. ER signal intensity varied between cells. Fig. 3 demonstrates an overlap (yellow) between ERα and ERβ with MitoTracker Red, indicating colocalization of each ER subtype within mitochondria of HBEC2-KT cells. Similar results were detected in HBEC2-E, HBEC3-ET, and HBEC4-KT cells (Supplemental Fig. 3). In contrast, ERα did not overlap with MitoTracker Red in HBEC3-KT (Supplemental Fig 3). Treatment of HBEC2-KT with 10 nM E2 for 45 min. did not alter the subcellular distribution of ERα and ERβ (Fig. 3) and similar results were detected with most HBECs (Supplemental Fig 3 and Table 2) with the exception that E2 appeared to decrease the nuclear and increase the cytoplasmic localization of ERα in HBEC3-ET (Supplemental Fig 3 and Table 2). This is the opposite of the effect of E2 in MCF-7 human breast cancer cells (Schlegel et al., 1999).

Fig. 3
ERα and ERβ localize to the cytoplasm, nucleus, and mitochondria in HBEC2-KT. Confocal microscopic imaging of HBEC2-KT treated with vehicle (EtOH) or 10 nM E2 for 45 min. Cells were incubated with 25 nM MitoTracker Red CMXRos (red color) ...
Table 2
Subcellular localization of ERα and ERβ in HBECs.

3.3. E2 and 4-OHT stimulate ERE-reporter gene activity in an HBEC cell line-specific manner

To determine the transcriptional activity of endogenous ER in the HBEC cell lines, transient transfection assays were performed using a luciferase reporter driven by 2 tandem copies of an ERE (Klinge et al., 2004). E2 increased ERE-driven luciferase activity in all HBECs (Fig. 4). 4-OHT and ICI 182,780 suppressed E2-induced transcription in HBEC2-E and HBEC3-ET, indicating that the increase in luciferase activity in response to E2 is ER-mediated. In HBEC2-E, the combination of E2 and 4-OHT reduced transcription to below basal, lower than either E2 or 4-OHT alone. 4-OHT did not inhibit E2- induced luciferase activity in HBEC2-KT, HBEC3-KT, or HBEC4-KT, although ICI inhibited E2-luciferase activity in these 3 HBEC cell lines, indicating that ER was responsible for the E2-induced activity. ICI inhibited basal luciferase activity in HBEC4-KT. 4-OHT showed ER agonist activity HBEC2-E and HBEC3-KT. It is well-established that 4-OHT is a selective ER modulator (SERM) that has ER agonist or agonist activity in a cell line-dependent manner (MacGregor and Jordan, 1998). In sum, these data indicate that endogenous ER is transcriptionally active in HBECs.

Fig. 4
Estradiol increases ERE-reporter gene expression in HBECs. HBECs were transiently transfected with an ERE-luciferase reporter as described in Section 2. The cells were treated with 10 or 100 nM E2, 100 nM 4-OHT, 1 μM ICI 182,780, alone or in combination ...

3.4. Transfection of HBECs with ERα-VP16 or ERβ-VP16 stimulated luciferase transcription

There are many individual steps in the ER signal transduction cascade at which defect(s) may reside. Because the ERα-VP16 and ERβ-VP16 chimeric proteins have the VP16 acid transcriptional activation domain (AD) cloned onto the N-terminus of the ER proteins, these constructs provide an assay of transcriptional activity that depends exclusively on DNA binding (McDonnell et al., 1995). Transfection with ERα-VP16 generated significantly higher ERE-driven luciferase activity in each HBEC cell line (Fig. 5). Most cell lines transfected with ERα-AF-1 showed only a modest increase in luciferase activity over basal, indicating that AF-1 is not predominant over AF-2 in HBECs, unlike HepG2, COS-7, or PC-3 cells (Flouriot et al., 2000; Merot et al., 2004; Metivier et al., 2002a,b; Penot et al., 2005). E2 did not result in a further increase reporter activity with VP16-ERα, possibly indicating that the transcriptional response is saturated, and actually inhibited VP16-ERα activity in HBEC4-KT. Likewise, E2 inhibited VP16-ERβ activity in HBEC3-ET and HBEC3-KT.

Fig. 5
Transfection of HBECs with ERα-VP16 or ERβ-VP16 increases ERE-driven luciferase activity. HBECs were transiently transfected with an ERE-luciferase reporter and 10ng VP16-ERα, VP16-ERβ, or ERα-AF-1 as described ...

3.5. E2 stimulates transcription of select endogenous estrogen-target genes in HBECs

Quantitative real time PCR (Q-PCR) was performed to examine the effect of E2 on the transcription of classical endogenous estrogen target gene cyclin D1 (CCND1) (1) and on the transcription of nuclear respiratory factor-1 (NRF-1, NRF1) which we recently identified as an estrogen-regulated gene (Mattingly et al., 2008). As controls, MCF-7 (ERα positive, estrogen-responsive), H1395 and H1792 (ERα and ERβ positive, E2-responsive (female) and non-responsive (male) lung adenocarcinoma cell lines, respectively (Dougherty et al., 2006)) were treated in parallel with HBECs. E2 increased CCND1 in MCF-7 and H1395 (Fig. 6), in agreement with the E2-induced proliferation in these cell lines (Dougherty et al., 2006). E2 increased CCND1 transcription in HBEC2-KT, HBEC3-ET, HBEC3-KT, and HBEC4-KT cells, and statistically reduced CCND1 expression in H1792 cells. Similarly, E2 increased NRF1 transcription in MCF-7, H1395, HBEC2-KT, HBEC3-ET, HBEC3-KT, and HBEC4-KT cells and reduced NRF1 in H1792 cells. In each cell line in which E2 increased CCND1 or NRF1 transcription, ICI inhibited the E2-induced increase, indicating that the transcriptional response was ER-mediated. Interestingly, ICI also reduced basal expression of CCND1 or NRF1 in each cell line, indicating that ER regulates the basal expression of these genes in these cell lines. In contrast, HBECs did not express two other classical E2-regulated target genes, i.e., PR and pS2, either in control or E2-treated cells (data not shown). Likewise, pS2 was not expressed in H1395 or H1792, but PR was E2-induced in H1395 (Supplemental Fig 6). These data indicate that E2 activates the transcription of some established estrogen target genes in HBECs.

Fig. 6
Estradiol increases endogenous estrogen target gene expression in HBECs. The indicated cell lines were treated with vehicle (EtOH, open bars), 10 nM E2 (closed bars), or 10 nM E2 + 100 nM ICI 182,780 (hatched bars) for 6 h. Q-RT-PCR analysis of cyclin ...

3.6. SMRTβ is the predominant SMRT isoform in HBECs

Although E2-induced ERE-luciferase activity in each of the transiently transfected HBECs (Fig. 4), E2 failed to increase cyclin D1 or NRF-1 in HBEC2-E. Coactivators are required for E2-ER induced gene transcription (Klinge, 2000). To determine whether differential expression of the coregulator SMRT corresponds with E2-induced CCND1 transcription in all HBECs except HBEC2-E (Fig. 6), we examined SMRT protein by Western blot using an antibody that recognizes both SMRTβ and SMRTβ (Fig. 7). We observed 2 bands corresponding to SMRT splice variants SMRTβ (full length) and SMRTβ which is missing repressor domain 1 (RD-1) at the N-terminus (Ordentlich et al., 1999). SMRTβ acts as an ERα-selective coactivator in a cell-type and gene-specific manner and is required for induction of CCND1 transcription and cell proliferation in MCF-7 cells (Peterson et al., 2007). SMRTβ expression was higher than SMRTα in HBECs and total SMRT expression was lower in HBECs compared to H1395, A549, and H1792 lung adenocarcinoma and MCF-7 breast cancer cells (Fig. 7). However, the inability of E2 to induce CCND1 transcription in HBEC2-E and H1792 and the lower induction of CCND1 transcription in HBEC3-ET and HBEC3-KT is not due to an absence or reduction of SMRTβ protein expression.

Fig. 7
SMRTβ expression is higher than SMRTα in HBECs. WCE (35 μg protein) from the indicated cell lines were separated by 3-8% Tris-Acetate gels, transferred to PVDF membranes, and probed with SMRT antibody as described in Section 2 ...

3.7. E2 stimulates and selective estrogen receptor modulators (SERMs) inhibit HBEC proliferation in a cell line-specific manner

E2 stimulated the proliferation of lung adenocarcinoma cell lines from female but not male patients (Dougherty et al., 2006). To determine if E2 affects HBEC proliferation in a gender-dependent manner, HBECs were treated with E2, 4-OHT, and ICI 182,780, alone or in combination with E2 and cell proliferation was measured by BrdU incorporation assays (Fig. 8). E2 increased proliferation of HBEC2-KT, HBEC3-KT, and HBEC4-KT, but not HBEC2-E or HBEC3-ET. The E2-dependent increase in HBEC3-KT and HBEC4-KT proliferation was inhibited by 4-OHT and ICI, indicating an ER-mediated effect. In contrast, 4-OHT and ICI did not inhibit the E2-induced proliferation in HBEC2-KT. Notably, the E2-induced proliferation of HBEC2-KT, HBEC3-KT, and HBEC4-KT correlates with the induction of Cyclin D1 transcription in these cells (Fig. 6). On the other hand, although E2 increased Cyclin D1 transcription in HBEC3-ET (Fig. 6A), it did not increase BrdU incorporation (Fig. 8).

Fig. 8
Estradiol increases the proliferation of HBEC2-KT and HBEC4-KT cells. The effect of the indicated concentrations of E2, 4-OHT, and ICI 182,780, alone or in combination with 10 nM E2 on the proliferation of HBEC cell lines was determined after 48 h of ...

4. Discussion

Although post-pubertal, premenopausal women have higher incidences of certain lung diseases, i.e., asthma (Carey et al., 2007a,b), and pre- and peri-menopausal are at increased risk for NSCLC, particularly adenocarcinoma (Schwartz et al., 2007), compared to men, few investigators have examined the effect of estrogens on normal bronchial epithelial cells. Only recently has estrogen been reported to act as a promoter for lung adenocarcinoma development in a genetically defined murine model (Hammoud et al., 2008). To our knowledge, the studies reported here are the first to systematically evaluate the expression, subcellular localization, and genomic function of ERα and ERβ in HBECs. Our results showing that all 5 HBEC cell lines express ~twice as much ERβ protein compared to ERα support results from IHC studies of human lung tissues (Pietras et al., 2005; Shen et al., 2007) and our previous report of higher ERβ than ERα in primary HBECs (Dougherty et al., 2006). Further, both ER subtypes were predominantly cytoplasmic and showed mitochondrial and nuclear staining. These data agree with other reports of mitochondrial localization of ERα and ERβ other cell types (Cammarata et al., 2004; Chen et al., 2007, 2004; Jonsson et al., 2007; Monje and Boland, 2001; Pedram et al., 2006; Solakidi et al., 2005; Yang et al., 2004).

Each of the HBEC lines used in our study have microarray gene expression profiles similar to primary HBECs and distinct from lung cancer cell lines (Ramirez et al., 2004). Here E2-induced ERE-driven luciferase activity in all HBECs, indicating that the endogenous ERs are transcriptionally activity. In contrast, E2 did not induce reporter activity in transiently transfected lung adenocarcinoma cell lines derived from male patients (Dougherty et al., 2006).

Here we observed that the E2-induced increase in cyclin D1 and NRF-1 transcription was correlated with E2-induced cell proliferation in HBEC2-KT, HBEC3-KT, and HBEC4-KT cells, but not in HBEC3-ET. CCND1 is amplified in NSCLC and frequently overex-pressed in lung tumors and pre-invasive bronchial lesions (Gautschi et al., 2007). However, to our knowledge, there is nothing published about ERα and cyclin D1 in normal lung. Increased cyclin D1 is associated with ERα expression in breast tumors (Cho et al., 2008; Stendahl et al., 2004) and CCND1 transcription is induced by E2 in MCF-7 and other ERα-expressing breast cancer cell lines (Altucci et al., 1996; Eeckhoute et al., 2006; Hong et al., 1998; Watts et al., 1994). Likewise, NRF1 transcription is induced by E2 in MCF-7 cells (Mattingly et al., 2008). NRF-1 is a transcription factor that regulates the transcription of mitochondrial transcription factors and genes involved in cell proliferation (Scarpulla, 2006). Although the NRF-1 transcript was highly expressed in rat lung (Gopalakrishnan and Scarpulla, 1995), this is the first study examining NRF-1 in normal human lung cells.

Although SMRT was originally characterized as a corepressor of ER and other nuclear receptors (Chen and Evans, 1995; Nagy et al., 1997), E2-ERα-induced cyclin D1 transcription in MCF-7 cells requires SMRTβ (Peterson et al., 2007). A recent editorial highlighted the importance of rethinking about coactivators and corepressors as context-specific coregulators having either stimulatory or inhibitory activities (O’Malley and McKenna, 2008). We observed no correlation between SMRTβ expression and E2-stimulated CCND1 transcription in the HBECs or in H1792 and H1395 lung adenocarcinoma cell lines. However, SMRTβ was higher than SMRTβ in HBECs. Since SMRTβ is missing RD-1, a hypothesis stemming from these data is that SMRT may play more of a coactivator than corepressor role in HBECs. Interestingly, only HBEC4-KT cells were growth inhibited by 4-OHT alone and HBEC4-KT showed higher SMRTα than the other HBECs, corresponding with the greater repressor role for SMRTα than SMRTβ (Peterson et al., 2007). Further experiments are needed to examine this suggestion. To our knowledge, this is the first examination of SMRT protein expression in HBECs or lung adenocarcinoma cells. Lung homogenates from male Sprague–Dawley rats had lower SMRT than those from females, but because the MW of SMRT was given as 160 kD (González-Arenas et al., 2004), the identity of this SMRT iso-form is unclear since SMRTα is 270 and SMRTβ is 249 kDa (Peterson et al., 2007). We did not detect a significant difference in SMRTβ expression in HBECs from females versus a male.

SMRT’s ERα-specific coactivator activity is cell-specific (Peterson et al., 2007). The coactivator activity of SMRT correlates with greater ERα AF-2 than AF-1 activity in MCF-7 and HeLa cells compared to HepG2 (Penot et al., 2005). Because E2 induced ERE-luciferase reporter activity in all HBECs (Fig. 5), and since we observed no increase in ERE reporter activity when HBECs were transfected with ERα-AF-1, a construct in which AF-2 is inactivated by 3 aa substitutions (Tzukerman et al., 1994), we conclude that ERα AF-2 > AF-1 in HBECs. Although 4-OHT was an agonist in HBEC2-E and HBEC3-KT, there is no apparent correlation of these responses to SMRTα or β expression.

There are few studies of ER activity in HBECs. A previous study reported no endogenous ERα at either the mRNA or protein levels in primary HBECs, but demonstrated that adenoviral ERα infection increased cigarette smoke extract-induced CYP1B1 transcription, a result that the authors interpreted as indicating that ERα contributes to gender differences in carcinogen metabolism and mutation (Han et al., 2005). The human bronchial epithelial cell line BEAS-2B, immortalized by SV40, expresses ERβ (Mollerup et al., 2002). E2 stimulates BEAS-2B cell proliferation by ~5% and E2 synergized with BaP to increase COX-2 transcription and PGE2 production in an ER-dependent manner (Chang et al., 2007). The authors speculated that BaP activated CYP1A1/CYP1B1 and increased catechol estrogen production thereby activating the NF-κB pathway in BEAS-2B cells (Chang et al., 2007). We have not examined E2 metabolism in the HBECs used in the present study.

In summary, although E2 stimulated transcriptional and proliferative activities in lung adenocarcinoma cell lines isolated from female but not male patients (Dougherty et al., 2006), in the present small study no gender-specific differences in the estrogenic pheno-type of HBECs was detected. Thus, there appears to be no innate gender-dependent difference in E2 responses in normal HBECs that correlate with those detected in the lung adenocarcinoma cell lines. Further, the estrogenic responses in lung adenocarcinoma cell lines appear to be greater than those in HBECs, implying a gain-of-function in the transformed cells.

Supplementary Material

01

Supplemental Fig. 1: N-terminal ERα antibodies detect ERα in HBECs. WCE (10 or 20 μg of MCF-7 and 40 μg of HBECs) from the indicated cell lines and the indicated amounts of baculovirus-expressed ERα were separated by 10% SDS PAGE, transferred to PVDF membranes, and probed with H184 (Santa Cruz cat. no. sc-7207, is a polyclonal rabbit antiserum raised against aa 2-185 of ERα) in panel A and IF3 (GeneTex cat. no. GTX70171, a monoclonal antibody raised against N-terminal aa 1-190 of ERα) in panel B. The membranes were stripped and re-probed for α-tubulin as a loading control for normalization as described in Materials and Methods. Migration of the MW standards is indicated at the left (kDa). (C) Summary of ERα expression from 4 separate western blots with the indicated ERα antibodies. Values are plotted as fmoles ERα/μg WCE protein and were calculated using the rhERα as a standard as described in Materials and Methods. Note the agreement in ERα levels in MCF-7 as detected with AER320, HC-20, H184, but not IF3.

Supplemental Fig. 2: HBECs express ERβ. WCE (40 μg for the PA1-311 (Affinity Bioreagents, raised against a peptide corresponding to aa 55-70 from rat ERβ) and 6A12 (GeneTex cat. no. GTX70179, raised against aa 1-153 of human ERβ) blots and 30 μg for the H150 blot) from the indicated cell lines and the indicated amounts of baculovirus-expressed ERβ were separated by 10% SDS PAGE, transferred to PVDF membranes, and probed with the indicated antibodies for ERβ. The membranes were stripped and re-probed for α-tubulin as a loading control for normalization as described in Materials and Methods. Migration of the MW standards is indicated at the left in kDa. The MW estimated by UnScanIt for rhERβ is indicated at the right in the top two blots (56 kDa).

Supplemental Fig. 3: ERα and ERβ localize to the cytoplasm, nucleus, and mitochondria in HBECs. Confocal microscopic images of the indicated HBEC cell lines and the gender of the patients from whom these cell lines were derived are indicated. Cells were treated with vehicle (EtOH) or 10 nM E2 for 45 min. Cells were incubated with 25 nM MitoTracker Red CMXRos (red color, bottom left quadrant in each block of four images) for 45 min prior to fixation. After fixation, the cells were incubated with ERα antibody HC-20 or ERβ antibody H-150 and then the secondary antibody labeled with Quantum Dot 525 (green color) as described in Materials and Methods. Cells were then incubated with ProLong® Gold anti-fade reagent with DAPI to stain the nucleus (blue color, upper left quadrant and in the merged image). Bars (gold) are 20μm. The merged image is in the lower right quadrant (Merge). The Figure at the bottom is a key to the 4 images in each block of four.

Supplemental Fig. 4: Demonstration of specificity of ER staining. Confocal microscopic imaging of MCF-7 cells incubated with 25 nM MitoTracker Red CMXRos (red color, bottom left quadrant) for 45 min prior to fixation. After fixation, the cells were incubated without primary antibody and then with secondary anti-rabbit antibody labeled with Quantum Dot 525 (green color). Cells were then incubated with ProLong® Gold anti-fade reagent with DAPI to stain the nucleus (blue color, upper left quadrant and in the merged image). Bars (gold) are 20μm. The merged image is in the lower right quadrant (Merge).

Supplemental Fig. 5: ERα western blot using HC-20 antibody. WCE (40 μg) from the indicated cell lines and the indicated amounts of baculovirus-expressed ERα were separated by 10% SDS PAGE, transferred to PVDF membranes, and probed with HC-20 (Santa Cruz sc-543) for ERα. To demonstrate the specificity of the 3 major ERα bands, in panel B as indicated in the blot at the right, the HC-20 blocking peptide (Santa Cruz) was incubated at a 2:1 ratio with HC-20 antibody at a 1:1000 dilution in 5% BSA-TBS-Tween for 1 h at room temperature prior to incubation with the PVDF membrane overnight at 4°C. Importantly, the specificity of the bands detected by HC-20 was demonstrated by preincubation with the HC-20 blocking peptide which completely ablated the appearance of all 3 major ERα bands in MCF-7 and rhERα. The membranes were stripped and re-probed for α-tubulin as a loading control for normalization as described in Materials and Methods. Migration of the MW standards is indicated in bold Arial font at the left edges of the blots and the estimated MW of ERα bands from UnScanIt are indicated in regular Times-New Roman font at the right in panel A.

Supplemental Fig. 6: E2 induces PR mRNA expression in H1395 cells. H1395 cells were treated with vehicle (EtOH) or 10nM E2 for 6 h. RNA was harvested and Q-PCR was performed using ABI Taqman primers and probes as described in Materials and Methods. Values are from one experiment performed in quadruplicate and are fold ± SEM. * Significantly different from EtOH values, p < 0.01.

Acknowledgements

This work was supported by grants from Joan’s Legacy Foundation, LUNGevity Foundation, and the Kentucky Lung Cancer Research Program to C.M.K. and by NIH P50CA70907 and DOD VITAL grants to J.D.M. We thank Lei Zhao and Susan M. Isaacs for performing some of the experiments included in this manuscript. We thank Dr. Barbara J. Clark for her comments to improve this manuscript.

Footnotes

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mce.2009.01.021.

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